Two main subject areas are to be studied in this proposal; I. The differentiated function of urothelium in vivo and II. The growth and differentiation of urothelium grown in vitro. During contraction-expansion cycles of mammalian urinary bladder a very large portion of luminal plasmalemma is translocated to and from the cytoplasm as plaques of membrane in discoidal vesicles. It has been proposed that the movement of the membrane plaques between cytoplasm and cell surface is passively mediated by a pulley-like arrangement of intermediate filaments attached to particles of the membrane plaques. This hypothesis will be tested by isolating luminal cells and studying the filamentous composition of these cells, and their ability to contract. In addition, a morphometric examination of plaque translocation following treatment with metabolic poisons will be carried out. In vitro studies will utilize an epithelial growth system which allows growth, stratification, differentiation, and maintenance of homogenous, adult rat transitional epithelium in primary, secondary or tertiary culture. Using this system we propose to: attempt to develop a define culture media, evaluate the role of cell density in plating efficiency, assess the the need of collagen or other porous substrates in stratification, examine the mechanism whereby urine effects differentiation of particulate luminal membranes, study permeability characteristics, and evaluate the role, if any, of cyclic AMP in growth, differentiation, and permeability. It is felt that an understanding of the processes and mechanisms governing normal function and differentiation of urothelium will be enhanced by the proposed studies.